Journal: Brain research

Article Title: Rodent models of TDP-43: Recent advances

doi: 10.1016/j.brainres.2012.04.031

Figure Lengend Snippet: Rodent models of wild type or ALS-linked mutant TDP-43.

Article Snippet: TDP-43 fragments [antibody used] , 25 and 35 kDa fragments (detergent-soluble) in 1–2 months and older mice [Proteintech 10782-2-AP, anti-body to TDP-43N-260aa] , , Some low molecular weight fragments in spinal cord homogenate and cytosolic fractions [Proteintech 10782-2-AP, antibody to TDP-43N-260aa] , Low molecular weight fragments in spinal cord homogenate and cytosolic fractions [Proteintech 10782-2-AP, antibody to TDP-43N-260aa] , 25 and 35 kDa fragments in brain and spinal cord extract from hemizygous and homozygous mice [Proteintech 12892-1-AP, antibody to TDP-43 260aa-C] , 25 and 35 kDa fragments in brain lysates from nontransgenic, hemizygous, and homozygous mice [Proteintech 12892-1-AP, antibody to TDP-43 260aa-C].

Techniques: Mutagenesis, Expressing, Staining, Molecular Weight

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: Top 20 significant genes coexpressed with C9orf72 on COXPRESdb.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Binding Assay, Sequencing, Glycoproteomics

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: COXPRESdb search indicates coexpression of AGTPBP1 with C9orf72. The genes coexpressed with C9orf72 were identified by database search on COXPRESdb, as listed in . Interactive network of top 20 genes coexpressed with C9orf72 was visualized on Cytoscape web. ( A ) Correlation profile of gene expression between C9orf72 ( x axis: probe ID 225919_s_at) and AGTPBP1 ( y axis: probe ID 204500_s_at). ( B ) Molecular network of top 20 genes coexpressed with C9orf72. The color of nodes indicates yellow (nuclear), green (cytosol), blue (mitochondria), white (plasma membrane), and grey (ambiguous), determined by WoLF localization prediction program. The interaction between C9orf72 and AGTPBP1 is highlighted by red ellipse.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Gene Expression, Clinical Proteomics, Membrane

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: Coexpression of AGTPBP1 and C9orf72 mRNA in human neural cells. The mRNA expression was studied by RT-PCR and qPCR in human neural tissues and cultured cells. The panels ( A – C ) represent RT-PCR of ( A ) AGTPBP1, ( B ) C9orf72, and ( C ) G3PDH. The lanes (1–10) indicate (1) the FC of the human CBR with inclusion of the reverse transcription (RT) step, (2) CBR without inclusion of the RT step, (3) astrocytes, (4) NP cells, (5) NTera2 teratocarcinoma-derived neurons (NTera2 N), (6) SK-N-SH neuroblastoma, (7) IMR-32 neuroblastoma, (8) U-373MG glioblastoma, (9) T98G glioblastoma, and (10) HMO6 immortalized microglia. The panels ( D, E ) represent qPCR of ( D ) AGTPBP1 and ( E ) C9orf72, standardized against the levels of G3PDH, serving as an internal control. The panel ( F ) indicates Pearson’s correlation between AGTPBP1 and C9orf72 mRNA expression levels in the set of eight human neural cells.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Reverse Transcription, Derivative Assay, Control

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: Molecular interaction of AGTPBP1 and C9orf72 in cultured cells. The full-length AGTPBP1 protein tagged with HA and the full-length C9orf72 protein tagged with Flag were coexpressed in HEK293 cells. The protein extract was processed for ( A ) IP with anti-Flag antibody followed by WB with anti-HA antibody or ( B ) IP with anti-HA antibody followed by WB with anti-C9orf72 antibody. The CP domain of AGTPBP1 tagged with Flag and the full-length C9orf72 protein tagged with Myc or EGFP were coexpressed in HEK293 cells. The protein extract was processed for ( C ) IP with anti-Myc antibody followed by WB with anti-Flag antibody or ( D ) IP with anti-Flag antibody followed by WB with anti-GFP antibody. The protein extract of SK-N-SH cells was processed for ( E ) IP with anti-C9orf72 antibody followed by WB with anti-AGTPBP1 antibody. The lanes represent (1) input control, (2) IP with the specific antibody, and (3) IP with normal IgG.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Cell Culture, Control

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: Coexpression of AGTPBP1 and C9orf72 in cultured cells and human brains. The vectors of EGFP-tagged AGTPBP1 and RFP-tagged C9orf72 were cotransfected in NSC-34 motor neurons. Coexpression of AGTPBP1 and C9orf72 was studied in the CA1 hippocampal region of human brains derived from normal control (NC) subjects by double-labeling IHC. The panels ( A – I ) represent ( A, D ) (A, D) NSC-34, AGTPBP1 (green), ( B, E ) NSC-34, C9orf72 (red), ( C, F ) merge of AGTPBP1 and C9orf72 labeled with DAPI, ( G ) CA1, AGTPBP1 (red), ( H ) CA1, C9orf72 (green), and ( I ) merge of AGTPBP1 and C9orf72 labeled with DAPI.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Cell Culture, Derivative Assay, Control, Labeling

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: IHC of AGTPBP1 and C9orf72 expression in human brains. The immunoreactivity for AGTPBP1 or C9orf72 was studied in the hippocampus CA4 region and the frontal cortex (FC) of NC and AD brains by IHC. The panels (A – D) indicate (A) NC, CA4, AGTPBP1, (B) NC, CA4, C9orf72, (C) NC, FC, AGTPBP1, and (D) AD, FC, AGTPBP1. Scale bar = 200 μm.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Expressing

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: AGTPBP1 and C9orf72 mRNA expression in human brains. The mRNA expression levels were studied by qPCR in human brain tissues derived from a reference of the human FC (REF), four NC, six ALS, four PD, and seven AD cases listed in . The expression levels were standardized against those of G3PDH, serving as an internal control: ( A ) AGTPBP1, ( B ) C9orf72, and ( C ) comparison between AD and non-AD groups. The panels indicate (left) AGTPBP1 and (right) C9orf72. ( D ) Pearson’s correlation between AGTPBP1 and C9orf72 mRNA expression levels.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Expressing, Derivative Assay, Control, Comparison

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: AGTPBP1 and C9orf72 protein expression in human brains. The protein expression levels were studied by WB in human brain tissues derived from four NC, six ALS, four PD, and seven AD cases listed in . The expression levels were standardized against those of ACTB, serving as an internal control. ( A ) WB of (a) AGTPBP1, (b) C9orf72, (c) ACTB, and (d) HSP60. ( B ) Comparison between AD and non-AD groups. The panels indicate (left) AGTPBP1 and (right) C9orf72. ( C ) Pearson’s correlation between AGTPBP1 and C9orf72 protein expression levels.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Expressing, Derivative Assay, Control, Comparison

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: Knockdown of AGTPBP1 in SK-N-SH cells. The siRNA product directed to AGTPBP1 (SI) or a negative control RNA (NEG) was introduced in SK-N-SH cells by using Lipofectamine (LPF) RNAiMax reagent, followed by processing for qPCR and WB analysis. The panels ( A , B ) represent qPCR of ( A ) AGTPBP1 and ( B ) C9orf72 standardized against the levels of G3PDH mRNA, while the panels ( C – E ) indicate WB of ( C ) AGTPBP1, ( D ) C9orf72, and ( E ) ACTB, serving as an internal control. The lanes (1–3) represent the protein extract isolated from the cells treated with (1) LPF alone, (2) NEG, or (3) SI.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Knockdown, Negative Control, Control, Isolation

Journal: Journal of Central Nervous System Disease

Article Title: Bioinformatics Data Mining Approach Suggests Coexpression of AGTPBP1 with an ALS-linked Gene C9orf72

doi: 10.4137/JCNSD.S24317

Figure Lengend Snippet: Overexpression of POU2F1 in SK-N-SH cells. The POU2F1 expression vector or the control vector expressing LacZ was transfected in SK-N-SH cells by using Lipofectamine (LPF) 2000 reagent, followed by processing for qPCR and WB analysis. The panels ( A, B ) represent qPCR of ( A ) AGTPBP1 and ( B ) C9orf72 standardized against the levels of G3PDH mRNA, while the panels ( C, D ) indicate WB of ( C ) POU2F1 and ( D ) HSP60, serving as an internal control. The lanes (1–3) represent the protein extract isolated from the cells treated with (1) LPF alone, or the cells transfected with the expression vector of (2) POU2F1 or (3) LacZ.

Article Snippet: Then, the tissue sections were incubated at 4 °C overnight with a rabbit polyclonal anti-AGTPBP1 antibody raised against a peptide spanning amino acid residues 837–1186 of the human AGTPBP1 protein (14067–1-AP; Proteintech), a rabbit polyclonal anti-human C9orf72 antibody raised against a peptide spanning amino acid residues 165–215 of the human C9orf72 protein (sc-138763; Santa Cruz Biotechnology), whose specificity was validated previously, or a mouse monoclonal anti-AGTPBP1 antibody raised against a recombinant protein fragment spanning amino acid residues 368–753 of the human AGTPBP1 (9A3; LifeSpan Biosciences).

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Transfection, Isolation

Journal: Journal of Applied Biological Chemistry

Article Title: The inhibitory effects of glabridin on human platelet aggregation and thrombus formation

doi: 10.3839/jabc.2023.060

Figure Lengend Snippet: Fig. 3 Glabridin’s effects on thromboxane A2 generation and cPLA2/p38 phosphorylation. (A) Glabridin’s effects on collagen-induced thromboxane A2 generation. (B) Glabridin’s effects on collagen-induced cPLA2/p38 phosphorylation. Data are expressed as the mean ± standard deviation (n =4). *p <0.05, **p <0.01 versus collagen-stimulated human platelets

Article Snippet: Phospho-IP3R, Phospho-VASP (Ser 157 and Ser239), Phospho-PI3K, Phospho-Akt (Ser 473 and Thr308), Phospho-GSK-3α/β, β-actin, phosphor-SYK, phosphor-p38, and phosphor-cytosolic phospholipase A2 (cPLA2) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Photoreceptor-specific knockout of Dync1h1 . ( A ) Schematic of mouse cytoplasmic dynein heavy chain (DYNC1H1, gene symbol Dync1h1 ). Locations of the truncation point ( red arrow ), six AAA motor domains ( blue ), and microtubule-binding domain ( green ) are indicated. Locations of previously identified mutations in mice and zebrafish are shown below the protein. Dync1h1 knockout results in truncation of DYNC1H1 in its tail domain and eliminates the motor domain. ( B ) Cytoplasmic dynein drawn schematically with heavy chain ( blue ). Adapted from Hoang et al. ( C ) loxP sites of the floxed Dync1h1 allele are located in introns 23 and 25. ( D ) Following Cre-induced recombination, the null allele loses exons 24 and 25. ( E ) PCR amplification using P10 retinal DNA as template and primers X23F and loxP-R. The amplicon at 500 bp shows Cre-induced recombination has occurred in the Dync1h1 F/F ;iCre75 retina, while amplicon at 1600 bp represents Dync1h1 F/F present in the inner retina. ( F ) Genotyping of Dync1h1 F/F and Dync1h1 F/+ alleles with primers loxP-F and loxP-R in intron 25. M, size markers; W, water control. ( G ) Genotyping of iCre75. +, tail DNA containing the iCre75 transgene. ( H ) Genotyping of Egfp-Cetn2. +, tail DNA containing the Egfp-Cetn2 transgene. ( I ) Genotyping of Prom1-ETCre mice.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Knock-Out, Binding Assay, Amplification

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Scotopic and photopic electroretinography. ( A – C ) Average scotopic a-wave amplitudes as a function of light intensity (−4.5 to 2.4 log cd s·m −2 ) at P16 (A), P21 (B), and P30 (C). The scotopic rod Dync1h1 − / − a-wave amplitude decreases by P21 and is nearly extinguished at P30. ( D ) Summary of peak amplitudes at 2.4 log cd s·m −2 ; average peak scotopic a-wave amplitude and standard deviations are indicated. ( E , F ) Average photopic b-wave amplitudes as a function of light intensity (−0.1 to 1.9 log cd s·m −2 ) at P21 (E) and P30 (F). Decline of the P30 photopic b-wave is probably caused by bystander cone degeneration following loss of rods in the rod knockout.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Knock-Out

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Immunohistochemistry with DYNC1H1 and rhodopsin (Rho) antibodies. ( A–D ) Representative control (rows A, C) and rod Dync1h1 −/− retinas (rows B, D) labeled with antibodies directed against DYNC1H1 (rows A, B) and rhodopsin (rows C, D) at P14, P16, P18, P21, and P30. DYNC1H1 is located in the photoreceptor IS and OPL (photoreceptor terminals and bipolar cell dendrites). The rod Dync1h1 − / − ONL starts to shrink at P16, and only cone nuclei remain at P30. Rhodopsin-containing rod OSs are severely shortened by P21 and absent by P30. Note, almost no rhodopsin accumulates in the ONL. Scale bar : 20 µm. ( E ) Comparison of the average ONL thickness of control and rod Dync1h1 −/− central retinas illustrates loss of rod photoreceptors between P16 and P30. Error bars show the standard deviation. * P < 0.05 (Student's t -test). ( F ) Comparison of the average combined length of rod OS + IS for P10 to P30 control and rod Dync1h1 −/− retinas shows loss of OS and IS in the absence of DYNC1H1. Error bars show the standard deviation. * P < 0.05 (Student's t -test). For E and F, the numbers of mice for control and rodKO, respectively, were P10, n = 5, 4; P12, n = 8, 3; P14, n = 5, 3; P16, n = 8, 3; P18, n = 3, 3; P21, n = 7, 3; P30, n = 10, 3.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Immunohistochemistry, Labeling, Standard Deviation

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Immunohistochemistry with PDE6 and RIBEYE antibodies. ( A–D ) Representative control (rows A, C) and rod Dync1h1 −/− retinas (rows B, D) labeled with antibodies directed against PDE6 (MOE; rows A, B) and RIBEYE (rows C, D). Rod and cone OS are shortened by P16 and lost by P30. RIBEYE expression in the ribbon synapses is lost between P16 and P30 with diminished expression evident by P21.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Immunohistochemistry, Labeling, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Tamoxifen-induced DYNC1H1 depletion in the adult retina. ( A–D ) tam Dync1h1 −/− (tamoxifen-induced KO, first column ), uninjected Dync1h1 F/F ;Prom1-CreER T2 ( second column ), tamoxifen-injected Dync1h1 F/F retinas ( third column ), and tamoxifen-injected Dync1h1 F/+ ;Prom1-CreER T2 ( fourth column ) harvested at 1wPTI (A), 2wPTI (B), 3wPTI (C), or 4wPTI (D). Cryosections were labeled with anti-DYNC1H1 ( red ) and contrasted with DAPI ( blue ). Uninjected Dync1h1 F/F ;Prom1-CreER T2 and injected Dync1h1 F/F or Dync1h1 F/+ ;Prom1-CreER T2 retinas all maintain normal IS and ONL thickness throughout the experiment. In tam Dync1h1 −/− (induced KO) retina, the ONL and IS shrank between 2wPTI and 3wPTI, but this shrinking was not statistically significant until 4wPTI. In retinas with efficient knockout of Dync1h1, ONL thinning increased in severity by 4wPTI. ( E ) Plot of average central ONL thickness at 1, 2, 3, and 4wPTI showing ONL thickness decreases in the tam Dync1h1 −/− from 2wPTI to 4wPTI. * P < 0.05 (Welch's ANOVA). ( F ) Plot of average combined OS + IS lengths shows that tam Dync1h1 −/− rod and cone OS shrink between 2wPTI and 4wPTI. * P < 0.05 (Welch's ANOVA). For E and F, the numbers of mice for control injected, control uninjected, and tam Dync1h1 −/− , respectively, were 1wPTI, n = 5, 5, 5; 2wPTI, n = 3, 3, 3; 3wPTI, n = 5, 5, 4; 4wPTI, n = 11, 5, 6.

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Injection, Labeling, Knock-Out

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Effect of tamoxifen-induced DYNC1H1 deletion on rhodopsin expression. ( A–D ) Representative immunohistochemistry from tam Dync1h1 −/− ( first column ), uninjected Dync1h1 F/F ;Prom1-CreER T2 ( second column ), tamoxifen-injected Dync1h1 F/F retinas ( third column ), and tamoxifen-injected Dync1h1 F/+ ;Prom1-CreER T2 ( fourth column ) harvested at 1wPTI (A), 2wPTI (B), 3wPTI (C), or 4wPTI (D) and probed with anti-rhodopsin ( red ). Nuclei were contrasted with DAPI ( blue ). tam Dync1h1 −/− retina reveals rod OS shortening and ONL thinning from 2 to 3wPTI. Rod OS shortening and ONL thinning increased in severity by 4wPTI, with minor rhodopsin expression in the ONL. ( E – G ) Average scotopic ERG traces at 1.4 log cd s·m −2 from control and tam Dync1h1 −/− at 2wPTI (E), 3wPTI (F), and 4wPTI (G). Scotopic responses decrease between 2 and 4 wPTI in the tam Dync1h1 −/− .

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Expressing, Immunohistochemistry, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Effect of tamoxifen-induced DYNC1H1 deletion on cone-opsin expression. ( A–D ) Representative immunohistochemistry from tam Dync1h1 −/− ( first column ), uninjected Dync1h1 F/F ;Prom1-CreER T2 ( second column ), tamoxifen-injected Dync1h1 F/F retinas ( third column ), and tamoxifen-injected Dync1h1 F/+ ;Prom1-CreER T2 ( fourth column ) harvested at 1wPTI (A), 2wPTI (B), 3wPTI (C), or 4wPTI (D) probed with anti–M-opsin ( red ). ONL indicated by DAPI ( blue ). tam Dync1h1 −/− retina reveals loss/shortening of cone OS from 2 to 3wPTI. While some M-opsin is seen in the ONL at 4wPTI, cone OSs are absent. ( E–G ) Average photopic ERG traces at 1.4 log cd s·m −2 from control and tam Dync1h1 −/− at 2wPTI (E), 3wPTI (F), and 4wPTI (G). Photopic responses decrease between 2wPTI and 4wPTI in the tam Dync1h1 −/− .

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Expressing, Immunohistochemistry, Injection

Journal: Investigative Ophthalmology & Visual Science

Article Title: Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors

doi: 10.1167/iovs.62.14.23

Figure Lengend Snippet: Summary of ERG responses as a function of intensity in tam Dync1h1 −/− retina. ( A–D ) Average scotopic a-wave amplitudes ( left column ) and photopic b-wave amplitudes ( right column ) as a function of light intensity shrink between 2wPTI and 4wPTI. Injected control ( blue ), uninjected control ( green ), and tamoxifen-induced knockout ( red ). At 1wPTI (A), average scotopic and photopic ERGs amplitudes did not differ significantly between induced knockout and control. At 2wPTI (B), the average scotopic a-wave amplitude was slightly reduced compared to controls. Photopic ERGs did not differ. At 3wPTI (C), induced KO shows reduced average ERG amplitudes for both scotopic a-wave and photopic b-wave, but this reduction was only statically significant for the photopic 1.4 log cd s·m −2 intensity. At 4wPTI (D), the reduction in average ERG amplitude for both scotopic a-wave and photopic b-wave for the induced KO was statically significant for most ERG flash intensities. * P < 0.05 (unbalanced two-factor ANOVA, Tukey's honestly significant difference criterion).

Article Snippet: Antibodies, dilutions, and sources follow: DYNC1H1 (dilution 1:250, 12345-1-AP; Proteintech, Proteintech Rosemont, IL), , rhodopsin (1:1000, A15093; Abclonal, Abclonal Woburn, MA), PDE6 (1:1000; MOE Cytosignal, Cytosignal IRVINE, CA), OPN1MW/OPN1LW (1:500, AB5405; Millipore Sigma, Millipore Sigma St. Louis, MO), , and CtBP2/RIBEYE (1:10,000, 612044; BD Biosciences, BD Biosciences Franklin Lakes, NJ).

Techniques: Injection, Knock-Out